Fludarabine- (C2- methylhydroxyphosphoramide)- [anti-IGF-1R]: Synthesis and Selectively "Targeted"Anti-Neoplastic Cytotoxicity against Pulmonary Adenocarcinoma (A549)

Abstract

Introduction: Many if not most conventional small molecular weight chemotherapeutics are highly potent against many forms of neoplastic disease. Unfortunately, majority of an administered dose unintentionally diffuses passively into normal tissues and healthy organ systems following intravenous administration. One strategy for both increasing potency and reducing dose-limited sequela is the selective "targeted" delivery of conventional chemotherapeutic agents.

Materials and methods: The fludarabine-(C₂- methylhydroxyphosphoramide)-[anti-IGF-1R] was synthesized by initially reacting fludarabine with a carbodiimide to form a fludarabine carbodiimide phosphate ester intermediate that was subsequently reacted with imidazole to create an amine-reactive fludarabine- (C₂-phosphorylimidazolide) intermediate. Monoclonal anti-IGF-1R immunoglobulin was combined with the amine-reactive fludarabine- (C₂-phosphorylimidazolide) intermediate resulting in the synthesis of covalent fludarabine-(C₂-methylhydroxyphosphoramide)- [anti-IGF-1R] immunochemotherapeutic. Residual fludarabine and un-reacted reagents were removed by serial microfiltration (MWCO 10,000) and monitored by analytical-scale HP-TLC. Retained IGF-1R binding-avidity of fludarabine-(C₂- methylhydroxyphosphoramide)-[anti-IGF-1R] was established by cell-ELISA using pulmonary adenocarcinoma cell (A549) which over-expresses IGF-1R and EGFR. Anti-neoplastic cytotoxic potency of fludarabine-(C₂-methylhydroxyphosphoramide)-[anti- IGF-1R] was determined against pulmonary adenocarcinoma (A549) using an MTT-based vitality stain methodology.

Results: The fludarabine molar-incorporation-index for fludarabine- (C₂-methylhydroxyphosphoramide)-[anti-IGF-R1] was 3.67:1 while non-covalently bound fludarabine was not detected by analytical scale HP-TLC following serial micro-filtration. Size-separation fludarabine-(C₂-methylhydroxyphosphoramide)-[anti- IGF-1R] by SDS-PAGE with chemo luminescent autoradiography detected only a single 150-kDa band. Cell-ELISA of fludarabine- (C₂-methylhydroxyphosphoramide)-[anti-IGF-1R] measuring total immunoglobulin bound to exterior surface membranes of pulmonary adenocarcinoma (A549) increased with elevations in immunoglobulin-equivalent concentrations of the covalent fludarabine immunochemotherapeutic. Between the fludarabine-equivalent concentrations of 10^-10 M and 10^-5 M both fludarabine-(C₂- methylhydroxyphosphoramide)-[anti-IGF-1R] and fludarabine had ex-vivo anti-neoplastic cytotoxic potency levels that increased rapidly between the fludarabine-equivalent concentrations of 10^-6 M and 10^-5 M where cancer cell death percentages increased from 24.4% to a maximum of 94.7% respectively.

Conclusion: The molecular design and organic chemistry reaction schemes were developed for synthesizing fludarabine-(C₂- methylhydroxyphosphoramide)-[anti-IGF-1R] which possessed both properties of selective "targeted" delivery and anti-neoplastic cytotoxic potency equivalent to fludarabine chemotherapeutic.

Keywords: Anti-neoplastic cytotoxicity; Cytotoxic potency; Pulmonary adenocarcinoma.

Figure 1

Organic Chemistry Reaction Scheme for Synthesis of Covalent Fludarabine-(C_2-methylhydroxyphosphoramide)-[anti-IGF-1R] Immunochemotherapeutic. Phase-I Reaction Scheme (Row Plate#1): reaction of the fludarabine C₂-mono-phosphate group with 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide to transiently form a reactive fludarabine carbodiimide phosphate ester intermediate complex; Phase-II Reaction Scheme (Row Plate II): rapid spontaneous conversion of the transient Phase I reactive intermediate to the Phase-II fludarabine-phosphorylimidazolide amine-reactive intermediate in the presence of imidazole. Phase-IIl Reaction Scheme (Row Plate III): covalent phosphoramide bond formation between the Phase-ll fludarabine-phosphorylimidazolide amine-reactive intermediate and μ-mononamine of lysine residues within the amino acid sequence of anti-IGF-1R monoclonal immunoglobulin resulting in the synthesis of a covalent fludarabine-(C₂-methylhydroxyphosphoramide)-[anti-IGF-1R] immunochemotherapeutic.

Figure 2

Characterization of the molecular weight profile for the covalent fludarabine-(C₂-methylhydroxyphosphoramide)-[anti-IGF-1R] immunochemotherapeutic relative to reference control anti-IGF-1R monoclonal immunoglobulin fractions and protein molecular weight standards. Legends: (Lane-1) murine anti-human EGFR monoclonal immunoglobulin; and (Lane-2) fludarabine-(C₂-methylhydroxyphosphoramide)-[anti-IGF-1R]. The covalent fludarabine immunochemotherapeutic and monoclonal immunoglobulin fractions were size-separated by non-reducing SDS-PAGE followed by lateral transfer onto sheets of nitrocellulose membrane to facilitate detection with HRPO-Protein G conjugate. Subsequent analysis entailed incubation with a HRPO chemiluminescent substrate and the acquisition of autoradiography images.

Figure 3

Evaluation of fludarabine-(C₂-methylhydroxyphosphoramide)-[anti-IGF-1R] by analytical HP-TLC for the detection of residual fludarabine not covalently bound to anti-IGF-1R immunoglobulin. Legends : (Lane-1) Phase-II fludarabine-phosphorylimidazolide amine-reactive intermediate; and (Lane-2) Phase-III covalent fludarabine-(C₂-methylhydroxyphosphoramide)-[anti-IGF-1R] immunochemotherapeutic following serial micro-filtration (MWCO = 10-kDa). Standardized fludarabine-equivalent concentrations of fludarabine-(C₂-methylhydroxyphosphoramide)-[anti-IGF-1R] and the fludarabine-phosphorylimidazolide amine-reactive intermediate were applied to HP-TLC plates (silica gel, 250 μm thickness, UV 254 nm indicator) and developed utilizing a propanol/ ethanol/H₂0 (17:5:5 v/v) mobile phase. Identification of any residual fludarabine or un-reacted fludarabine-phosphorylimidazolide in the Phase-III covalent fludarabine-(C₂-methylhydroxyphosphoramide)-[anti-IGF-1R] immunochemotherapeutic was subsequently determined by direct illumination with UV light.

Figure 4

Detection of total immunoglobulin in the form of fludarabine- (C₂-methylhydroxyphosphoramide)-[anti-IGF-1R] selectively bound to the exterior surface membrane of pulmonary adenocarcinoma. Covalent fludarabine-(C₂-methylhydroxyphosphoramide)-[anti-IGF-1R] immunochemotherapeutic formulated at gradient immunoglobulin-equivalent concentrations were incubated in direct contact with triplicate monolayer populations of chemotherapeutic-resistant human pulmonary adenocarcinoma (A549) over a 4-hour time period. Total immunoglobulin bound to the exterior surface membrane was then detected and measured by cell-ELISA.

Figure 5

Relative cytotoxic anti-neoplastic potency of fludarabine- (C₂-methylhydroxyphosphoramide) - [anti - IGF-1R] against chemotherapeutic-resistant pulmonary adenocarcinoma. Legends: (α) fludarabine-(C2-methylhydroxyphosphoramide)-[anti-IGF-1R]; and fludarabine chemotherapeutic. Formulated in triplicate at gradient standardized (fludarabine-equivalent) concentrations, both fludarabine- (C₂-methylhydroxyphosphoramide)-[anti-IGF-1R] and fludarabine were separately incubated in direct contact with monolayer populations of chemotherapeutic-resistant pulmonary adenocarcinoma (A549) for a period of 192-hours. Cytotoxic anti-neoplastic potency was measured using a MTT cell vitality assay relative to matched negative reference controls.